Volume 10, Issue 18, Pages 1108-1117 (September 2000) Synaptic targeting and localization of Discs-large is a stepwise process controlled by different domains of the protein  U. Thomas, S. Ebitsch, M. Gorczyca, Y.H. Koh, C.D. Hough, D. Woods, E.D. Gundelfinger, V. Budnik  Current Biology  Volume 10, Issue 18, Pages 1108-1117 (September 2000) DOI: 10.1016/S0960-9822(00)00696-5

Fig. 1 DLG expression at body wall muscles of wild-type and UAS–DLG-expressing flies, and dlg1P20 mutants. The third instar body wall muscles shown were stained with antibodies directed against (a,c,d) DLG (anti-DLGPDZ antibody) or (b) the FLAGepitope tag (anti-FLAG antibody). (a) In wild-type flies, endogenous DLG immunoreactivity was concentrated at the NMJ, with weak expression at extrasynaptic regions (arrow). In flies expressing (c) UAS–DLG and (b) UAS–DLG–FLAG, DLG was concentrated at the NMJ, but was also prominent at extrasynaptic regions. (d) In dlg1P20 mutants, in which the last 40 carboxy-terminal amino acids of DLG have been deleted, expression of endogenous and extrasynaptic DLG was similar to that in wild-type flies. All the confocal images in this figure were acquired using the same confocal parameters. The scale bar represents 17μm. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 2 Developmental expression of synaptic and extrasynaptic DLG at body wall muscles of embryos. (a–c) Stage 16 (a) embryonic nervous system and (b,c) body wall muscles labeled with (a,b) anti-DLGPDZ or (c) anti-horseradish peroxidase (HRP) antibodies. (b,c) Same views of a preparation that was double-labeled. At this stage, DLG was concentrated in the central nervous system, and absent from muscles and synaptic terminals. (d–f) Stage 17 embryonic body wall muscles stained with anti-DLGPDZ antibody. (d,e) Single confocal slices taken (d) at the surface of the muscles and (e) at the level of the muscle nuclei. (f) Same view as (e), but double-stained with anti-HRP antibody to visualize the presynaptic aspect of the NMJ (arrow). Insets in (d,e) are high-magnification views of the extrasynaptic signal. At this stage of development, DLG appeared (f) at the presynaptic region, as well as (d) in a punctate pattern at the muscle surface and (e) in an intracellular network. The scale bar represents 28μm, except in the insets where it represents 7μm. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 3 Developmental expression of synaptic and extrasynaptic DLG localization at body wall muscles of larvae. Anti-DLGPDZ antibody immunoreactivity at body wall muscles of (a,b) first, (c,d) second, and (e,f) third instar larvae. (a,c,e) Single confocal slices at the surface of the muscles. (b,d,f) Single confocal slices at the level of the muscle nuclei. Insets in (a,b) are high-magnification views of extrasynaptic DLG. In first instar larvae, extrasynaptic DLG was still very prominent both at the muscle surface and intracellular network. Extrasynaptic immunoreactivity was very weak in the third instar stage. All confocal images were acquired using the same confocal parameters. The scale bar represents 28μm, except in the insets where it represents 7μm. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 4 Expression of FLAG-tagged DLG constructs. (a) Diagrammatic representation of the control (DLG–FLAG) and deletion constructs. The top diagram is a representation of the modular structure of DLG. The numbers in each line represent the amino acids immediately adjacent to the deletions. N, amino terminus; C, carboxyl terminus. (b) Western blot analyses of body wall muscle extracts from larvae expressing DLG constructs. Left, anti-DLGPDZ antibodies were used to detect the transgenic proteins in a dlgX1-2 mutant background. Right, anti-FLAG antibody was used to detect those variants that lacked partially or completely the epitopes for anti-DLGPDZ antibody, that is, the PDZ1 and 2 domains. Asterisks represent extracts of dlgX1-2 body wall muscles expressing the deletion constructs. All other lanes represent wild-type body wall muscle extracts expressing the deletion constructs. Numbers on the left represent molecular weight in kDa. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 5 Localization of DLG–FLAG deletion constructs in body wall muscles when expressed in a wild-type background. Left and middle columns are single confocal slices at the surface (left) and at the level of muscle nuclei (middle) of third instar body wall muscles stained with anti-FLAG antibodies. The right column is a diagrammatic representation of the distribution of FLAG immunoreactivity at different muscle sites, depicted at right angles to the plane of section of the micrographs. N, nucleus; SR, sarcoplasmic reticulum; SSR, subsynaptic reticulum; b, bouton; m, membrane. Listed on the right of the column are the names of constructs with similar patterns of FLAG distribution. (a) FLAG immunoreactivity in the DLG–FLAG control, which was similar to the immunoreactivity pattern observed for ΔN, ΔPDZ1, ΔPDZ2, ΔPDZ3, ΔPDZ1+3, ΔPDZ2+3, and ΔSH3. The arrowhead points to a segment of the immunoreactive subcortical network. (b) Localization of ΔPDZ1+2 to membrane-associated ectopic clusters (arrowhead). Note the severe reduction of immunoreactivity in boutons and subcortical area. (c) Nuclear, synaptic, cytoplasmic and membrane localization of ΔHOOK, which was similar to the localization of ΔE–F. (d) Weak synaptic and membrane localization of ΔGUK, which was similar to ΔI3 localization. (e) Nuclear and cytoplasmic localization of ΔC1/2. All confocal images were acquired using the same parameters. The scale bar represents 40μm. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 6 Distribution of endogenous DLG in flies expressing the ΔPDZ1+2 variant. (a) Anti-DLGGUK antibody immunoreactivity in dlgX1-2 body wall muscles expressing ΔPDZ1+2. (b) Anti-DLGPDZ antibody immunoreactivity in the same preparation as in (a). Note that the anti-DLGPDZ antibody used (which was directed against the PDZ1 and 2 domains) does not recognize the ΔPDZ1+2 protein. (c) Anti-DLGGUK antibody immunoreactivity in wild-type flies expressing ΔPDZ1+2. Both endogenous and transgenic protein were labeled by this antibody. (d) Anti-DLGPDZ antibody immunoreactivity in the same preparation as in (c), showing that the presence of extrasynaptic clusters to which ΔPDZ1+2 was localized did not affect the distribution of endogenous DLG. The scale bar represents 42μm. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 7 FLAG localization in dlgX1-2 mutants expressing (a) DLG–FLAG, (b) ΔGUK, and (c) ΔI3. Unlike the situation in the wild type, the ΔGUK protein failed to localize at synapses in the mutant background, whereas the ΔI3 protein showed an increased localization to synapses. The scale bar represents 35μm. Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)

Fig. 8 DLG targeting to synapses is a stepwise process that requires multiple domains. The deletion analysis supports a model in which the HOOK region mediates direct or indirect association of DLG with an intracellular membrane compartment (1), presumably the sarcoplasmic reticulum, from which DLG is transported to the muscle plasma membrane (2 and 3). This transport may involve the association of vesicle-bound DLG with microtubules and/or a motor protein, as has been suggested for other MAGUKs [22,40]. In a process that requires the PDZ1 and 2 domains, DLG is then recruited to the subsynaptic reticulum (SSR). Current Biology 2000 10, 1108-1117DOI: (10.1016/S0960-9822(00)00696-5)